Isoelectric focusing is an electrophoretic technique wherein an electric field is applied to a molecule in a pH gradient to mobilize the molecule to a position in the pH gradient at which its net charge is zero, i.e., the isoelectric point of the molecule. It often is used to separate proteins in a mixture and as an aid in characterization of biomolecules of unknown composition. Commercially available gradients may be utilized in isoelectric focusing which consist of multicharged ampholytes, with closely spaced pI values and high conductivity, which partition into a pH gradient upon application of an electric field. The ampholytes are generally provided in a support matrix, such as a polyacrylamide gel. Molecules separated by isoelectric focusing may be visualized, e.g., by silver staining or Coomassie blue staining. Deutscher, Ed., Methods in Enzymology, Vol. 182, Academic Press, Inc., San Diego, Calif., 1990, Chapter 35.
Capillaries have been used in various electrophoretic techniques including isoelectric focusing. Novotny et al., Electrophoresis, 11:735-749 (1990). U.S. Pat. No. 5,061,361 (1991) relates to a capillary electrophoresis system in which a nanoliter volume of sample is introduced into the capillary tube, and an electric field is imposed on the system to effect separation of the charged components. After migration along the length of the tube, the sample components are detected via ultra-violet absorbance. U.S. Pat. No. 5,084,150 (1992) relates to an electrokinetic separation in which the surface of moving charged colloidal particles is treated so as to interact selectively with the sample molecules to be separated. An electric field is imposed on a capillary tube containing the colloidal particles and the sample to achieve separation. U.S. Pat. No. 5,045,172 (1991) relates to a capillary electrophoresis apparatus in which electrodes are attached at each end of a capillary tube, and a detector is coupled to the tube. U.S. Pat.. No. 4,181,589 (1980) relates to a method for separating biological cells using an electric field.
A wide range of specific binding assays, such as immunoassays, have been described in the prior art (see Bolton et al., Handbook of Experimental Immunology, Weir, D. M., ed., Blackwell Scientific Publications, Oxford, 1986, Vol. 1, Chapter 26, for a general discussion on immunoassays.) Antibody-antigen complexes have been resolved by isoelectric focusing in the prior art. Nielsen et al., J. Chromatography, 539:177-185 (1991). Japanese Patent Application No. 57026751 discloses the detection of an antigen-antibody complex using isoelectric focusing, wherein either the antigen or the antibody is chemically bound to a solid particle.